Biofouling of nanofiltration (NF) membranes is a major impediment in wastewater reclamation. However, research on the fouling process, including conditioning and subsequent biofouling in complicated systems, is limited. In this study, the combined effects of organic matter (OM) and calcium on Pseudomonas aeruginosa-induced fouling are systematically investigated and verified through an analysis of permeate flux, foulants, and membrane surface properties (roughness, surface charge, hydrophobicity). Sodium alginate (SA), bovine serum albumin (BSA), and humic acid (HA) are selected as model organics for polysaccharides, proteins, and humic substances in wastewater, respectively. Results show that approximately 8% of permeability is lost during organic-free conditioning in the absence and presence of Ca2+. However, subsequent biofouling is reduced at 5 and 8 mM Ca2+. In the presence of OM, Ca2+ plays an important role in organic conditioning and subsequent biofouling. SA and HA accelerate organic conditioning with the increase in Ca2+ concentration but inhibit subsequent biofouling. By contrast, severe biofouling occurs in the presence of BSA at 2 mM Ca2+, as revealed by both flux decline and the biomass accumulation. Organic conditioning significantly influences membrane surface properties and results in biomass retention on hydrophobic and rough surfaces conditioned with BSA. (C) 2015 Elsevier B.V. All rights reserved.